Vpr enhances an early stage of viral replication at the 2nd round of infection. (A) Schematic illustration of the rescue of viral replication assay by Vpr in target cells. Indicated cells were infected with VSV-G pseudotyped ΔVpr HIV-1 (NL4-3) by spinoculation. Three days later, virions were collected from supernatants, and equal amounts of virions were used to infect the HIV indicator TZM-b1 cells, which were transfected with a Vpr expression or control vector. The Vpr expression in TZM-b1 cells were determined by Western blotting (B), and viral replication in TZM-b1 cells was determined by measuring the intracellular luciferase activity (C). (D) To analyze viral protein expression, indicated cells were infected VSV-G pseudotyped WT or ΔVpr HIV-1 by spinoculation. Two days later, virions were purified from the culture supernatants by ultracentrifugation. Viral protein expression in infected cells and virions was detected by Western blotting. (E) To analyze viral early and late reverse transcription (RT), T4 cells were infected with WT or ΔVpr HIV-1 purified from spinoculated N5-P, N8-SP, and T4 cells, and 12 hours later, viral early and late RT products were quantitated by real-time PCR. Copy numbers were presented as relative values after normalization with mitochondrial DNA. The standard errors (S.E.) in (C) and (E) were calculated from three independent experiments.